Objective: To analyze the interactions between different structural types of volatile oil components (VOCs) and skin lipid molecules, and investigate the mechanism of volatile oil in Chinese materia medica (VOCMM) as penetration enhancers. Methods: In this study, 210 different structural types of VOCs were selected from the VOCMM penetration enhancer database, and the molecular docking experiments were conducted with three main lipid molecules of skin: ceramide 2 (CER2), cholesterol (CHL), and free fatty acid (FFA). Each VOC was docked individually with each lipid molecule. Cluster analysis was used to explore the relationship between the binding energy of VOCs and their molecular structures. Nine specific pathogen-free (SPF) Sprague Dawley (SD) rats were randomly divided into Control, Nootkatone, and 3-Butylidenephthalide groups for in vitro percutaneous experiments, with three rats in each group. The donor pool solutions were 3% gastrodin, 3% gastrodin + 3% nootkatone, and 3% gastrodin + 3% 3-butylidenephthalide, respectively. The penetration enhancing effects of VOCs with higher binding energy were evaluated by comparing the 12-hour cumulative percutaneous absorption of gastrodin (Q12, µg/cm²). Result: (i) Most of the VOCs were non-hydrogen bonded to the hydrophobic parts of CHL and FFA, and hydrogen bonded to the head group of CER2. Among them, sesquiterpene oxides showed the most pronounced binding affinity to CER2. The VOCs with 2 − 4 rings (including carbon rings, benzene rings, and heterocycles) demonstrated stronger binding affinity for three skin lipid molecules compared with the VOCs without intramolecular rings (P < 0.01). (ii) According to the cluster analysis, most of the VOCs that bond well to CER2 had 2 − 3 intramolecular rings. The non-oxygenated VOCs were bonded to CER2 in a hydrophobic manner. The oxygenated VOCs were mostly bonded to CER2 by hydrogen bonding. (iii) The results of Franz diffusion cell experiment showed that the Q12 of Control group was 260.60 ± 25.09 µg/cm2, and the transdermal absorption of gastrodin was significantly increased in Nootkatone group (Q12 = 5 503.00 ± 1 080.00 µg/cm², P < 0.01).  The transdermal absorption of gastrodin was also increased in 3-Butylidenephthalide  group (Q12 = 495.40 ± 56.98 µg/cm², P > 0.05). (iv) The type of oxygen-containing functional groups in VOCs was also an influencing factor of binding affinity to CER2. Conclusion The interactions between different types of VOCs with different structures in the VOCMM and three skin lipid molecules in the stratum corneum were investigated at the molecular level in this paper. This research provided theoretical guidance and data support for the screening of volatile oil-based penetration enhancers,  and a simple and rapid method for studying the penetration-enhancing mechanism of volatile oils.